Gene encoding 2,3-dihydroxybenzoic acid decarboxylase

ABSTRACT

The enzyme 2,3-dihydroxybenzoic acid decarboxylase has industrial applications for the decarboxylation of ring mounted carboxyls, specifically the decarboxylation of 2,3-dihydroxybenzoic acid to catechol. This invention relates to the isolation of a nucleic acid sequence from  Aspergillus niger  that encodes an enzyme that decarboxylates 2,3-dihydroxybenzoic acid. This invention further discloses the nucleic acid sequence, the protein sequence, vectors comprising the nucleic acid sequence, cells transformed with the nucleic acid sequence, and methods for the production of 2,3-dihydroxybenzoic acid decarboxylase.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/136,073,filed Aug. 18, 1998, now U.S. Pat. No. 6,043,076, which claims thebenefit of U.S. Provisional Application Ser. No. 60/056,621, which wasfiled with the U.S. Patent and Trademark Office on Aug. 20, 1997.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

This invention relates to the isolation of a nucleic acid sequence thatencodes an enzyme capable of removing carboxyl groups from aromaticrings. In particular the enzyme decarboxylates 2,3-dihydroxybenzoic acidto form catechol.

The decarboxylation reaction involves the non-oxidative removal of acarboxyl group from an aromatic ring. Aromatic rings containing acarboxyl group are chemically challenging to work with because thecarboxyl group is a deactivating group. Deactivating groups makeelectrophilic substitutions on aromatic rings difficult. Therefore, theremoval of a deactivating carboxyl group from an aromatic ring has agreat deal of potential in the chemical industry.

Catechol is an aromatic compound utilized in the development ofpharmaceuticals such as L-DOPA(L-3,4-dihydroxyphenylalanine) andadrenaline, agrobiochemicals such as carbofuran, and antioxidants suchas 4-tert-butyl catechol and veratrol. Additionally, catechol isutilized in the production of flavorants such as vanilla andpolymerization inhibitors. The current global noncaptive market forcatechol is 20.5×10⁶ Kg/yr.

Current commercial production of aromatics has several drawbacks. Onedisadvantage relates to the starting material utilized in aromaticproduction. Most aromatics are synthesized from benzene, toluene andxylene which are derived from petroleum or natural gas fossil fuelfeedstocks. For example, catechol is currently produced by distillationof coal tar or the hydroxylation of phenol. Both of these methodsrequire fossil fuels as starting material. Fossil fuels are nonrenewableand therefore more expensive than renewable resources. In addition, manycountries do not have a large national supply of fossil fuels for thederivation of aromatic compounds.

In addition to cost, the use of fossil fuels has a negative impact onthe environment. Petroleum based processes for the production ofaromatics often require hazardous starting materials. One example of ahazardous starting material is benzene, which is a carcinogen. Theseprocesses also produce hazardous waste by-products that caninadvertently leak into the environment. Hazardous waste by-productsalso need to be disposed of or stored, adding to the costs of operation.

Another problem with current aromatic compound production is that thesynthetic procedures involve harsh reaction conditions. Current methodsfor the removal of carboxyl groups from aromatic rings involve hightemperatures and acid conditions in the presence of metal catalysts.These extreme reaction conditions are expensive energy consumingprocedures that pose industrial and environmental safety concerns anddramatically increase the cost of the aromatics.

Therefore, a method for the production of aromatics, and specificallycatechol, is needed to overcome the problems associated with currentmethods utilized in the production of aromatics.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide analternative method for the removal of carboxyl groups form aromaticrings. In particular, it is an object of the present invention toprovide a method for the decarboxylation of 2,3-dihydroxybenzoic acid toform catechol. Specifically, the invention provides an isolated nucleicacid sequence encoding a stable enzyme that will catalyze thenon-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catechol.

Another object of the present invention is to provide an isolatednucleic acid sequence encoding a protein that catalyzes thenon-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catecholand that hybridizes, under stringent conditions, to SEQ ID NO:1. SEQ IDNO:1 comprises a nucleic acid sequence that encodes Aspergillus niger2,3-dihydroxybenzoic acid decarboxylase.

Yet another object of the present invention is to provide an isolatednucleic acid sequence encoding a protein that catalyzes thenon-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catecholand that hybridizes under stringent conditions to a nucleic acidsequence corresponding to an amino acid sequence of SEQ ID NO:2.

A further object of the present invention is to provide fragments of thenucleic acid sequence encoding 2,3-dihydroxybenzoic acid decarboxylasethat hybridize to SEQ ID NO: 1 and that code for products that maintainbiological activity necessary to decarboxylate 2,3-dihydroxybenzoicacid. These fragments can be either recombinant or synthetic or acombination thereof.

A further object of the present invention is to provide a recombinantvector comprising a nucleic acid sequence encoding a protein thatcatalyzes the non-oxidative decarboxylation of 2,3-dihydroxybenzoic acidto catechol. The definition of a vector for the purposes of thisinvention is any nucleic acid sequence into which a foreign nucleic acidsequence may be inserted wherein the nucleic acid sequence containingthe foreign nucleic acid sequence may be used to introduce the foreignnucleic acid sequence into a host cell. This vector may also compriseregulatory elements operably linked to the nucleic acid sequence.

A further object of the present invention is to provide various cellstransformed with the vector comprising a nucleic acid sequence encodinga protein that catalyzes the non-oxidative decarboxylation of2,3-dihydroxybenzoic acid to catechol.

A further object of the present invention is to provide methodology forthe production of the various products of the present invention.Examples include isolated nucleic acid sequences encoding2,3-dihydroxybenzoic acid decarboxylase, isolated 2,3-dihydroxybenzoicacid decarboxylase, isolated SEQ ID NO:1, isolated SEQ ID NO:2, and theprotein product of SEQ. ID. NO.:1.

A further object of the present invention is to illustrate the use of anenzyme for the synthesis of catechol and the decarboxylation of aromaticacids. This approach reduces environmental concerns associated withtraditional methods for the production of aromatics.

2,3-dihydroxybenzoic acid decarboxylase of the present invention issuited to accomplish these and other related objects of the presentinvention by catalyzing a reaction whereby a carboxyl group is removedfrom an aromatic ring. Specifically, a carboxyl group is removed from2,3-dihydroxybenzoic acid to produce catechol and carbon dioxide. Thisdecarboxylation is unusual in that it involves a non-oxidativedecarboxylation from an aromatic nucleus and does not require acofactor. This enzyme is found in the fungus Aspergillus niger andfunctions in the pathway for the metabolism of indole. This enzyme islisted in the Enzyme Commission by the no. EC.4.1.1.46

Aspergillus niger is a known fungus and is readily available to thosewith ordinary skill in the art. Additionally, Aspergillus niger has beendeposited with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209. The deposit was filed withATCC on Aug. 10, 1998. The culture is identified as Aspergillus nigerCSVInd and by the ATCC Accession No. 74460. Additionally, thepET22b(+)DHBD vector, as described in this specification, has beendeposited with the ATCC, 10801 University Blvd., Manassas, Va.20110-2209. The deposit was filed with ATCC on Aug. 10, 1998. The vectoris identified as pET22b(+)DHBD and by the ATCC Accession No. 203104.These deposited materials are available pursuant to all requirements ofthe United States Patent and Trademark Office.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows and in part willbe apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawing forms a part of this specification and is to beread in conjunction therewith.

FIG. 1 illustrates the decarboxylation reaction whereby2,3-dihydroxybenzoic acid decarboxylase catalyzes the decarboxylation of2,3-dihydroxybenzoic acid to catechol.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

An isolated nucleic acid sequence encoding 2,3-dihydroxybenzoic aciddecarboxylase is disclosed. Additionally, synthetic oligonucleotidesused in isolating the sequence encoding 2,3-dihydroxybenzoic aciddecarboxylase are disclosed.

Nucleic acid sequences presented by this disclosure will enable thecreation of full-length nucleic acid molecules encoding2,3-dihydroxybenzoic acid decarboxylase and fragments thereof.Specifically, the disclosed oligonucleotides of SEQ. ID. NOs. 3-5, orany other sequences that hybridize to the ends of SEQ.ID.NO.1, may beutilized to produce copies of the disclosed nucleic acid sequence whichmay be used for the expression of the disclosed protein. Additionally,it is well-known by those of ordinary skill in the art thatoligonucleotide design allows an unlimited choice for incorporatingrestriction endonuclease sites into a vector and a product. Theflexibility in oligonucleotide selection and restriction endonucleasesite selection allows several choices for the method of preparation ofthe nucleic acid sequences of the present invention.

It is also well-known to practitioners of the art that the nucleic acidsequences may be recombinant or synthetic or partly synthetic and partlyrecombinant. Recombinant implies the use of molecular biology tools.Synthetic refers to the use of chemical synthetic procedures.

Isolated products of the present invention are disclosed. These productsinclude but are not limited to nucleic acids, peptides and proteins. Theterm “nucleic acid” includes both DNA and RNA sequences. Some examplesof DNA and RNA sequences include cDNA and mRNA, respectively. An exampleof a protein product is the translated protein product of SEQ.ID.NO. 1.

The present invention discloses vectors containing the nucleic acidsequences encoding 2,3-dihydroxybenzoic acid decarboxylase. In oneembodiment, the vector includes regulatory sequences operably positionedwithin the vector, whereby the nucleic acid sequences encoding2,3-dihydroxybenzoic acid decarboxylase are expressed. These vectorsused for expressing a given protein, or a portion thereof, are commonlyreferred to as expression vectors. Often, these expression vectorscomprise at least one origin of replication, at least one promoter, atleast one ribosome binding site and at least one terminator. Vectors mayoften contain many other sequence elements known to those of ordinaryskill in the art. These vectors may be of viral, procaryotic oreukaryotic origin and may serve a variety of functions not limited toexpression.

The present invention discloses cells transformed with various vectors.In one embodiment, this cell is an E. coli cell of the appropriategenetic makeup, transformed with an expression vector of the presentinvention is capable of expressing 2,3-dihydroxybenzoic aciddecarboxylase. Such cells, commonly known as host cells, can be eitherprocaryotic or eukaryotic. It is understood by those of ordinary skillin the art that a recombinant molecule containing the nucleic acidsequences of the present invention can be used to transform a variety ofhosts using any known technique for transformation. Additionally, thereare other methods besides transformation for the introduction of nucleicacids into host cells. These methods include, but are not limited to,transfection and direct introduction of nucleic acid sequences.

In addition to the nucleic acid sequences, vectors, and transformedcells, the present invention further discloses a method for theproduction of 2,3-dihydroxybenzoic acid decarboxylase. This methodinvolves the insertion of a nucleic acid sequence that encodes2,3-dihydroxybenzoic acid decarboxylase into an expression vector. Thisvector may then be transformed into an appropriate host bacterial cellso that the nucleic acid sequences encoding 2,3-dihydroxybenzoic aciddecarboxylase may be expressed. The 2,3-dihydroxybenzoic aciddecarboxylase is then isolated from the bacteria through proteinpurification techniques. Through this process, 2,3-dihydroxybenzoic aciddecarboxylase can be produced in large quantities for use in industryand research. Additionally, it is known to those skilled in the art thatthere are several methods besides transformation with expression vectorsthat allow the expression of protein from a given nucleic acid sequence.One example is transfection of certain host cells with RNA. In anotherexample, expression may be achieved from a nucleic acid sequence invitro rather than in vivo. Host cells containing a nucleic acid sequenceof interest can, in some instances, be used directly to carry out thereaction of interest. Thus, transformed cells may be used to directlycatalyze the conversion of 2,3-dihydroxybenzoic acid decarboxylase tocatechol.

The present invention discloses the product of the above-discussedmethod. This product is an isolated protein that decarboxylates2,3-dihydroxybenzoic acid. This isolated product can be used in industryto catalyze the formation of catechol from 2,3-dihydroxybenzoic acid.

The present invention discloses a method for removing a ring mountedcarboxyl group by non-oxidative decarboxylation. This is accomplished bycausing a host cell to produce a protein having a biological activitysubstantially equal to 2,3-dihydroxybenzoic acid decarboxylase whereinsaid protein is encoded by a first nucleic acid sequence that hybridizesto a second nucleic acid sequence comprising SEQ ID NO: 1 understringent conditions. This protein is then combined with moleculeshaving a ring mounted carboxyl group and allowed to react underdecarboxylation conditions to accomplish the non-oxidativedecarboxylation.

The present invention discloses isolated nucleic acid sequences thatencode 2,3-dihydroxybenzoic acid decarboxylase. The procedures listedbelow may be implemented to produce large quantities of the disclosednucleic acid sequences. Additionally, these sequences can be producedthrough either synthetic or recombinant means. These procedures allowthe industrial and scientific community access to the nucleic acidsequence of the present invention for further studies or for the purposeof expression in order to produce and isolate 2,3-dihydroxybenzoic aciddecarboxylase.

Characterization of the Nucleic Acid Sequence Encoding2,3-Dihydroxybenzoic Acid Decarboxylase

The recombinant nucleic acid sequence, SEQ ID NO: 1, consists of an openreading frame of 1029 nucleotides. The start codon is at ATG and thestop codon at TAG. The conceptual translation product of this openreading frame is a protein, 342 amino acids in length. The length of 342amino acids is as expected for a subunit size of 38,000 that has beendetermined by SDS-PAGE for the wild type enzyme protein from Aspergillusniger. The calculated molecular weight of the translated product is39155.45 Da. This is similar to the molecular weight of 39,162 Dadetermined by mass spectrometric measurements for the wild type enzymeprotein of Aspergillus niger. All partial sequences determined for thisprotein by amino acid sequencing, have been identified in this openreading frame. These partial sequences are illustrated in Santha et al.,Eur.J.Biochem, 230, p. 104-110, 1995, which is hereby incorporated byreference. In particular, the N-terminal sequence is identical to thatdetermined for the wild type protein from Aspergillus niger.Additionally, the sequence of the last four amino acids at theC-terminus of the protein was determined through C-terminal sequencingof the native protein from Aspergillus niger. The identical amino acidsequence was discovered at the end of the reading frame in therecombinant nucleic acid sequence. The identity of three residues in therest of the protein is different between the translated sequence and theprotein sequence determination. These differences are, in someinstances, due to errors in reading of sequencing results and, in someinstances, due to the ambiguity in the determination of Asp and Cys onthe protein sequencing machine used for the purpose. Another feature tonote is that the translated product of the recombinant nucleic acidsequence contains the active site peptide with the Cys at the expectedposition as described in the above reference. This confirmed that theinsert contained the coding sequence for 2,3-dihydroxybenzoic aciddecarboxylase.

Characterization of Recombinant 2,3-Dihydroxybenzoic Acid Decarboxylase

Recombinant 2,3-dihydroxybenzoic acid decarboxylase is comparable to thewild type, or naturally occurring, Aspergillus niger2,3-dihydroxybenzoic acid decarboxylase. For example, both enzymescatalyze the conversion of 2,3-dihydroxybenzoic acid to catechol.Neither the recombinant nor the wild type requires an externally addedcofactor or metal ions for activity. The recombinant enzyme, like thewild type, is very stable.

Sequence comparisons were performed to determine the similarities in thesequences between the wild type and the recombinant. The recombinantprotein was sequenced from the N-terminus and the first 20 amino acidswere determined. This sequence was identical to that of the wild typeprotein.

The molecular weight of the wild type and the recombinant were comparedon SDS gels. The recombinant protein and the wild type protein fromAspergillus niger comigrate on SDS gels at approximately 38,000 Da.Therefore, the subunit molecular weight of the recombinant enzyme issimilar to the wild type at 38,000 Da.

The kinetic constants for both the wild type enzyme and the recombinantenzyme are similar. The recombinant enzyme has a K_(m) of 0.35 mM whichis comparable to a K_(m) of 0.43 mM for the wild type enzyme fromAspergillus niger. The ¹³C kinetic isotope effect, KIE, for the wildtype enzyme was 1.031±0.001 at the optimum pH of 5.2, and under similarconditions, the KIE for the recombinant enzyme was 1.033, as reported inSantha et al., FASEB J, 11, p A1017, abstract #932, 1997, posterpresented at the 17^(th) IUBMB Congress, San Francisco, which is herebyincorporated by reference.

The immunochemical cross reactivity was compared between the recombinantand the wild type from Aspergillus niger. Antibodies raised in rabbit tothe wild type protein from Aspergillus niger successfully identify therecombinant protein during standard western blot procedures.

Additional Characteristics of Wild Type 2,3-Dihydroxybenzoic AcidDecarboxylase from Aspergillus niger

2,3-dihydroxybenzoic acid decarboxylase operates in the fungal pathwayfor the degradation of indole in Aspergillus niger. Its existence hasalso been demonstrated in Aspergillus oryzae and Trichosporon cutaneum.The enzyme non-oxidatively decarboxylates 2,3-dihydroxybenzoic acid toform catechol and carbon dioxide. The enzyme does not require anyexternally added cofactor or metal ions for its activity. The enzyme wasassayed spectrophotometrically by following the disappearance of2,3-dihydroxybenzoic acid at 305 nm and by the appearance of catechol at276 nm. The K_(m) of the enzyme for 2,3-dihydroxybenzoic acid is 0.43 mMat a pH of 5.2. The enzyme appears to be specific for2,3-dihydroxybenzoic acid and does not appear to decarboxylate salicylicacid(2-hydroxybenzoic), 2,4-dihydroxybenzoic acid or3,4-dihydroxybenzoic acid.

The native molecular weight (M_(r)) for 2,3-dihydroxybenzoic aciddecarboxylase as assessed by gel filtration on a Sephacryl S-200 columnis 150,000 Da. SDS gels of the protein illustrate a single band of38,000 Da. Thus, the protein is a homotetramer of subunit sizeapproximately 38,000 Da.

2,3-dihydroxybenzoic acid decarboxylase has an optimal activity at a pHof 5.2. Substantial activity (80-90%) is found in a range between pH 5and 5.5. In addition, the enzyme is stable to at least a pH of 7.5.

The temperature for optimal activity of the enzyme increases withtemperature to 50° C., after which the activity begins to decrease dueto denaturation of the protein. Additionally, the enzyme is stable attemperatures above 50° C. in the presence of substrate analogs. Oneexample of a substrate analog is salicylate. Assays were routinely doneat 30° C. Enzyme purification was done at 4° C.

It has been found that certain modifying reagents, such as histidine,cysteine and tryptophan inactivate 2,3-dihydroxybenzoic aciddecarboxylase. This inactivation may be due to the modification of oneor more amino acids at or near the active site of the enzyme.

The following discussion will assist in defining the structure for aportion of the claimed invention. The genetic code is degenerate. Thisis because there are several combinations of three nucleotides that willcode for the same amino acid. For example, Leu can be coded for by 6combinations of 3 nucleotides. Many nucleic acid sequences can code fora particular amino acid sequence. Thus, there is a multitude of nucleicacid sequences that could code for SEQ ID NO:2. It is intended that atleast those nucleic acid sequences that both hybridize under stringentconditions with the nucleic acid sequence comprising SEQ ID NO: 1 andcode for apeptide having substantially the same biological activity as2,3-dihydroxybenzoic acid decarboxylase are included within the scope ofthis invention.

Stringent conditions are defined as hybridization in a medium containing6×SSC or 6×SSPE and 40% formamide at a temperature of 37° C. and a washat 37° C. with 2×SSC or 2×SSPE containing 0.1% SDS. These solutions wereprepared from 20×SSC or SSPE which comprise the following compositions.The 20×SSC is formed by mixing 175.3 g of sodium chloride and 88.2 g ofsodium citrate in 1 L of water with a final pH of 7.0. The 20×SSPE isformed by mixing 175.3 g of sodium chloride and 88.2 g of sodium citratein 1 L of water with a final pH of 7.4. Any nucleic acid sequences thathybridize to SEQ.ID.NO. 1 in a solution containing 6×SSC or 6×SSPE and40% formamide at 37° C. and remains bound to SEQ.ID.NO. 1 when themilieu is changed to 2×SSC or 2×SSPE containing 0.1% SDS are thosenucleic acid sequences defined as hybridizing under stringentconditions. Additionally, it is well known in the art that severalfactors affect hybridization including the length and nature of theprobe, nature of the target, concentrations of salts, components in thehybridization solution, and temperature. Therefore, this description ofstringency includes equivalent hybridization and wash conditions.

In describing the nucleic acid sequences of the present invention, bothstructure and function are utilized. The structure is provided by theabove description of stringency; those nucleic acid sequences thathybridize under stringent conditions. The function is provided by theabove discussion of the biological activity of 2,3-dihydroxybenzoic aciddecarboxylase. Therefore, those nucleic acid sequences that hybridize toSEQ.ID.NO.: 1 and whose protein products have activity similar to2,3-dihydroxybenzoic acid decarboxylase are included within the scope ofthe present invention. Additionally, those nucleic acid sequences thathybridize to SEQ.ID.NO.: 1 and whose protein products have activitysimilar to 2,3-dihydroxybenzoic acid decarboxylase may code for peptidefragments and are also included within the scope of the presentinvention.

Those of ordinary skill in the art will recognize that methods forvector construction and protein expression provided in the followingexamples are the preferred embodiment and that there are othertechniques, vectors, and cell lines that could be implemented forconstructing and expressing proteins or fragments thereof in eitherprocaryotic or eukaryotic systems. The preferred embodiment disclosedherein does not limit the scope of the invention. There are a variety ofalternative techniques and procedures available to those with ordinaryskill in the art that would permit one to perform modifications on thepresent invention. It is also well known in the art that commerciallyavailable kits allow the modification and incorporation of the presentinvention. It is further recognized that those with ordinary skill inthe art could employ any of a number of known techniques to modify thenucleic acid sequences of the present invention, in vitro or in vivo,and develop them further by established protocols for gene transfer andexpression.

Several advantages are achieved by utilizing the present invention. Forexample, one advantage is that recombinant technology allows for theproduction of large quantities of 2,3-dihydroxybenzoic aciddecarboxylase for commercial and scientific use.

Another advantage realized by the present invention is that it offers anenzyme that is capable of decarboxylating aromatic acids without the aidof any cofactor. This feature of non-requirement for a cofactor is anadvantage when compared to traditional chemical procedures that requiremetals.

Another advantage realized by the present invention is that it offersenzyme based chemical procedures. Enzyme based chemical procedures areenvironment friendly and cost effective unlike typical chemicalprocedures that are associated with environmental concerns.

Another advantage realized by the present invention is that2,3-dihydroxybenzoic acid decarboxylase may be utilized with otherprocedures for the production of catechol. This combination ofprocedures will create alternate methods for the synthesis of catecholthat will not depend on fossil fuels.

EXAMPLE I Growth of Aspergillus niger

Aspergillus niger was grown on Byrde's modified synthetic mediumcontaining anthranilic acid which served as an inducer of2,3-dihydroxybenzoic acid decarboxylase as well as a nitrogen source forthe growth of the organism, as illustrated by Karnath et. al., Appliedand Environmental Microbiology, January 1990, p. 275-280, which ishereby incorporated by reference. The medium composition per liter ofwater was as follows: glucose (5 g), anthranilic acid (2 g), potassiumdihydrogen phosphate (KH₂PO₄, 5 g), magnesium sulfate (MgSO₄.7H₂O, 1 g),sodium sulfate (Na₂SO₄, anhydrous, 1 g) and trace element mixture (5 mlconsisting of FeCl₃.6H₂O, 20 mg; ZnSO4.10H₂O, 10 mg; MnSO₄.4H₂O, 3 mg;Na₂MoO₄.2H₂O, 1.5 mg and CuSO₄.5H₂O, 0.1 mg). Trace elements were addedfrom a 200× stock after the anthranilic acid had dissolved completely.The pH of the medium was adjusted to 5.5 with 3NNaOH. Sterile mediumcontained in Erlenmeyer flasks, 10% of the total flask volume, wasinoculated with a dense spore suspension and the cultures left to growunder stationary conditions at 30 ° C. White mycelial felts wereharvested at 24-26 hours after inoculation. The spent medium wasdecanted off. The mycelia were washed thoroughly in distilled water,dried between folds of Whatman paper 3MM and frozen in liquid nitrogen.Frozen mycelia were stored at −70° C. until further use for theisolation of RNA.

EXAMPLE II Isolation of a Gene for 2,3-Dihydroxybenzoic AcidDecarboxylase

Isolation of mRNA

Total Aspergillus niger RNA was isolated from the frozen samples ofExample I. These samples are ground in liquid nitrogen and the total RNAextracted utilizing the TRIzol™ reagent described in the LIFETECHNOLOGIES™ protocol, revision date Dec. 22, 1993, which is herebyincorporated by reference. mRNA was isolated from the total RNAutilizing the procedures provided with FAST TRACK® 2.0 kit fromINVITROGEN®. FAST TRACK® 2.0 kit manual version B, 160228 is herebyincorporated by reference.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) of mRNA

The mRNA was reverse transcribed to generate fall-length first strandcDNA molecules using the procedures provided with the cDNA CYCLE® kitfor RT-PCR from INVITROGEN®. The manual for the cDNA CYCLE kit versionA, 150525 is hereby incorporated by reference. Features that relate tothe experiment described here are the use of 1 μg of mRNA and the use ofoligonucleotide represented in SEQ ID NO:3 in place of the oligo dTprovided with the kit. The oligonucleotide of SEQ ID NO:3 is also anoligo dT primer, but has additional bases that represent a Not Irestriction endonuclease site. This site was used in later cloningprocedures.

The products of the reverse transcriptase reaction were utilized astemplates in a PCR reaction designed to specifically amplify the nucleicacid sequence corresponding to 2,3-dihydroxybenzoic acid decarboxylase.This was accomplished by the use of a degenerate oligonucleotide primerconstructed to represent the coding sequence of the N-terminus of2,3-dihydroxybenzoic acid decarboxylase from, Aspergillus niger. Thisoligonucleotide is represented by SEQ.ID.NO. 4. The sequence of theN-terminus is illustrated in Santha et al., Eur.J.Biochem, 230, p.104-110, 1995, which is hereby incorporated by reference. For theconstruction of the primer, a codon usage table for the genusAspergillus was created using the Genetics Computer Group (GCG) sequenceanalysis package. The codon frequencies for the amino acids at the firstsix positions of the N-terminus were evaluated to determine the bestchoice of degenerate oligonucleotides to represent the N-terminus.Degenerate first or third bases were used at positions with codonfrequencies equal to or lesser than 0.5. The primer ended with the firsttwo, instead of three bases for Ala, in order to provide a 3′ GC ‘clamp’for priming. In addition to this degenerate sequence, SEQ.ID.NO.4comprised of bases at the 5′ end that represent the EcoRI restrictionendonuclease site. This site was used in cloning procedures.

The N-terminal specific oligonucleotide represented by SEQ.ID.NO.4 wasused along with the oligo dT primer represented by SEQ.ID.NO.3 toamplify the products of the reverse transcriptase reaction using Taqpolymerase. Conditions for the PCR reaction were as follows: 100 ngreverse transcribed product from 100 ng of starting mRNA, 10 μl PROMEGA®10×PCR buffer mix (without Mg²⁺), 15 pmol SEQ.ID.NO.3, 15 pmolSEQ.ID.NO.4, 0.5 U Taq polymerase from PROMEGA), 2 mM Mg²⁺, 0.2 mM dNTPsand H₂O in a final reaction volume of 100 μl. The reaction mix withoutthe Mg²⁺ and the dNTPs was incubated at 80° C. for 1 min before startingthe reaction by the addition of the Mg²⁺-dNTP mix. The PCR cycling wasperformed in two stages. The first stage consisted of 5 cycles ofdenaturation at 94° C for 1 minute, annealing at 45 ° C. for 1 min andextension at 72 ° C. for 1 minute. The second stage consisted of 25cycles of denaturation at 94 ° C. for 1 minute, annealing at 64° C. for1 minute and extension at 72° C. for 1 minute. The cycling reaction wasallowed to proceed to completion by a further incubation at 72° C. for 7minutes. An agarose gel analysis revealed that a single product of size1.1-1.2 kb was formed. This product was gel purified using theGENECLEANS® kit from Bio 101. The protocol for the GENECLEAN® Kit,Revision No.1001-699-5F01, is hereby incorporated by reference.

Cloning of PCR Product

The PCR amplified nucleic acid sequences were then cloned into a pCR™IIvector utilizing the procedures provided with a TA CLONING® Kit fromINVITROGEN®. The TA CLONING® Kit, Version B, 150626, is herebyincorporated by reference. Ligation products or plasmid DNA used in thisexperiment, and at all stages throughout this study, were isolatedaccording to established procedures. These procedures includetransformation into a host such as E. coli DH5αF′ cells, TOP10F′ orINVαF′ cells from INVITROGEN® or Novablue cells from NOVAGEN® andpreparation of the plasmid using the NUCLEOBOND® kit from CLONTECH®.Procedures for transformation are outlined in the TA CLONING® Kit,Version B, 150626 from INVITROGEN and the pET System Manual, 6thEdition, TB055 8/95, provided by NOVAGEN®, respectively. Both thesemanuals and the NUCLEOBOND® nucleic acid purification tools user manual,PT3167-1 (PR84333), are hereby incorporated by reference. Plasmids weretested for insert size. Several positive clones were obtained and thesewere sequenced. Sequencing of these and several other clones describedhereafter, was carried out at the DNA Sequencing Core Facility at theUniversity of Nebraska-Lincoln Center for Biotechnology. Automated DNAsequencing was performed on LI-COR Model 4000 and LI-COR Model 4000L DNAsequencers using fluorescent primers in the dideoxy chain terminationmethod. The sequences of the inserts, in all clones, contained thesequence described in SEQ ID NO. 1. In addition, there was an Eco RIrestriction site before the start codon and a Not I site after the polyA stretch. These vectors were designated as pTA-DHBD-EcoRI.

EXAMPLE III Expression of the Gene for 2,3-Dihydroxybenzoic AcidDecarboxylase

Preparation of the Insert for Cloning into pET22b(+)

A nucleic acid insert containing SEQ.ID.NO.1 was generated frompTA-DHBD-EcoRI. This insert contained a Nde I restriction site locatedat the initial ATG instead of the EcoRI restriction site found inpTA-DHBD-EcoRI. The insert was generated through PCR with pTA-DHBD-EcoRIas a template, and oligonucleotides represented in SEQ.ID.NO. 3 andSEQ.ID.NO.5 as primers. Reaction conditions were the same as describedabove for RT-PCR of mRNA. A single product of size 1.1-1.2 kb wasobtained. The insert was purified from a gel using the GENECLEANO® kitfrom Bio 101 as discussed above. The purified product was cloned into aTA vector, pCR™II vector of INVITROGEN® using the TA CLONING® kit fromINVITROGEN® as discussed above. Several positive clones with inserts ofthe proper size and sequence were obtained. The insert sequence for thisvector contains a sequence identical to SEQ.ID.NO.:1. Additionally, theinsert had an Nde I site overlapping the initial ATG and a Not I siteafter the poly A tail. This vector was designated pTA-DHBD-NdeI.

Cloning into the Expression Vector pET22b(+)

The insert from pTA-DHBD-Nde I, which contained a sequence identical toSEQ.ID.NO. 1, was released from the vector by enzymatic digestion withrestriction endonucleases Nde I and Not I. The insert was gel purifiedby the GENECLEAN® method referenced above. Purified inserts were ligatedand cloned in a pET22b(+) vector that was subjected to enzymaticdigestion with the same restriction endonucleases, Nde I and Not I. Theresulting vector contained the insert for 2,3-dihydroxybenzoic aciddecarboxylase positioned at the Nde I site. This positioning ensuredthat the start codon ATG was positioned at an appropriate distance fromthe T7 promoter and that the coding sequence was in the correct readingframe for the protein to be expressed. This vector was designatedpET22b(+)DHBD.

Expression of Recombinant 2,3-Dihydroxybenzoic Acid Decarboxylase

Expression of 2,3-dihydroxybenzoic acid decarboxylase was achieved bytransforming pET22b(+)DHBD into an E.coli expression host, BL21(DE3).The procedures for transformation and expression were performedaccording to the pET System Manual, 6th Edition, provided by NOVAGEN®,TB055 8/95, which is also covered by U.S. Pat. No. 4,952,496. Both themanual and the patent are hereby incorporated by reference. Transformedcells were grown at 37° C. in Luria Broth medium containing ampicillinand chloramphenicol to an absorbance at 600 nm of 0.6-1.0. Cells wereinduced with isopropyl-β-D-thiogalactoside (IPTG) at a concentration of0.4 mM and allowed to incubate with shaking at 37° C. for another 3hours at which point the cells were harvested. The harvested cells werestored at −20° C. These cells were used for the isolation of2,3-dihydroxybenzoic acid decarboxylase.

EXAMPLE IV Isolation of Recombinant 2,3-Dihydroxybenzoic AcidDecarboxylase

Cells induced for expression of 2,3-dihydroxybenzoic acid decarboxylase,as described above in Example III, were suspended in an equal volume of50 mM sodium phosphate buffer, pH 7.0, containing protease inhibitors.These protease inhibitors included pepstatin (10 μg/ml), leupeptin (10μg/ml), phenylmethanesulfonyl fluoride (1 mM), 1,10-phenanthroline (5mM) and EDTA (1 mM). The suspension was sonicated in 3 pulsed cycles at2 minute intervals. The 2,3-dihydroxybenzoic acid decarboxylase waspurified from this lysate using a combination of ion exchangechromatography and affinity chromatography. Purified preparations weredetermined to be homogenous by SDS-PAGE and N-terminal sequencing. Theabove procedure is described by Santha et. al., Biochimica et BiophysicaActa, 1293, 1996, p. 191-200, and references therein. This publicationis hereby incorporated by reference.

EXAMPLE V Preparation of Multiple Copies of Vectors Containing Inserts

Multiple copies of vector containing insert, for example, pET22b(+)DHBD,were prepared by transformation of Novablue® cells as described in thepET System Manual, 6th Edition, TB055 8/95, provided by NOVAGEN®, whichis hereby incorporated by reference. Multiple copies of pTA-DHBD-EcoRIor pTA-DHBD-Nde I, were prepared by transformation of TOP10F′ or INVαF′®cells as described in the TA CLONING® kit manual, Version B, 150626 fromINVITROGEN, which is hereby incorporated by reference.

All references discussed herein are specifically incorporated in theirentirety in all respects.

From the foregoing, it may be seen that this invention is onewell-adapted to achieve all the ends and objects hereinabove set forthtogether with other advantages which are obvious and which are inherentto the invention. It will be understood that certain features andsubcombinations are of utility and may be employed without reference toother features and subcombinations. The above examples discuss thetechniques and procedures utilized and are considered to be thepreferred embodiment of the current invention, and it is understood thatthere are many other techniques and procedures that could be employedwhich would allow an individual of ordinary skill in the art to performthe claimed invention. Such other techniques and procedures arecontemplated by and are within the scope of the claims. Since manypossible embodiments may be made of the invention without departing formthe scope thereof, it is to be understood that all matter herein setforth and shown in the drawings and examples are to be interpreted asillustrative and not in a limiting sense.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211>LENGTH: 1096 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220>FEATURE: <221> NAME/KEY: gene <222> LOCATION: 1-1096 <221> NAME/KEY: CDS<222> LOCATION: 1-1029 <221> NAME/KEY: polyA_site <222> LOCATION: 1073<221> NAME/KEY: polyA_signal <222> LOCATION: 1056-1061 <221> NAME/KEY:3′ UTR <222> LOCATION: 1027-1073 <221> NAME/KEY: source/Aspergillusniger <222> LOCATION: 1-1096 <300> PUBLICATION INFORMATION: <301>AUTHORS: Santha, Ramakrishnan Dickman, Martin B. O′Leary, Marion H.<302> TITLE: 2,3-Dihydroxybenzoic Acid Decarboxylase From Aspergillusniger: Mechanism, Cloning And Overexpression. <303> JOURNAL: FasebJournal <304> VOLUME: 11 <305> ISSUE: 9 <306> PAGES: A1017 <307> DATE:1997-07-31 <400> SEQUENCE: 1 atg ttg ggt aag atc gcc ctc gaa gaa gcc ttcgcg ctt ccc cgc ttc 48 Met Leu Gly Lys Ile Ala Leu Glu Glu Ala Phe AlaLeu Pro Arg Phe 1 5 10 15 gaa gag aag aca cgc tgg tgg gcc agt cta ttctcc gtc gac ccc gaa 96 Glu Glu Lys Thr Arg Trp Trp Ala Ser Leu Phe SerVal Asp Pro Glu 20 25 30 acc cac gtc aag gag atc acc gac atc aac aag ctgcgc atc gaa cat 144 Thr His Val Lys Glu Ile Thr Asp Ile Asn Lys Leu ArgIle Glu His 35 40 45 gcc gac aag tac ggc gtg gga tac cag atc ctc tcc tacaca gct ccc 192 Ala Asp Lys Tyr Gly Val Gly Tyr Gln Ile Leu Ser Tyr ThrAla Pro 50 55 60 ggt gtc caa gac att tgg gat ccc gtt gaa gcc caa gcc ctagcc gtt 240 Gly Val Gln Asp Ile Trp Asp Pro Val Glu Ala Gln Ala Leu AlaVal 65 70 75 80 gaa atc aac gac tac atc gca gag cag atc cgc gac aag cccgat cgc 288 Glu Ile Asn Asp Tyr Ile Ala Glu Gln Ile Arg Asp Lys Pro AspArg 85 90 95 ttt ggc gca ttt gca acc ctc tcc atg cac aac ccc caa gaa gccgcc 336 Phe Gly Ala Phe Ala Thr Leu Ser Met His Asn Pro Gln Glu Ala Ala100 105 110 tct gag ctc cgc cgc tgc gtc caa acc tac ggc ttc aaa ggc gcccta 384 Ser Glu Leu Arg Arg Cys Val Gln Thr Tyr Gly Phe Lys Gly Ala Leu115 120 125 gta aac gac acc caa cgc gcc ggc ccc gac ggc gac gac atg atcttc 432 Val Asn Asp Thr Gln Arg Ala Gly Pro Asp Gly Asp Asp Met Ile Phe130 135 140 tac gac aac gcc tcc tgg gat atc ttc tgg caa aca tgc acg gaactc 480 Tyr Asp Asn Ala Ser Trp Asp Ile Phe Trp Gln Thr Cys Thr Glu Leu145 150 155 160 gac gtc cct ctg tac ttg cac cct cgc aac ccc acc ggc accatc tac 528 Asp Val Pro Leu Tyr Leu His Pro Arg Asn Pro Thr Gly Thr IleTyr 165 170 175 gag aag ctc tgg gca gac cgg aaa tgg ctc gtg ggt ccg ccgctc agc 576 Glu Lys Leu Trp Ala Asp Arg Lys Trp Leu Val Gly Pro Pro LeuSer 180 185 190 ttc gcg cag ggc gtc agt ctg cac gtt ctg ggg atg gtc acgaac ggc 624 Phe Ala Gln Gly Val Ser Leu His Val Leu Gly Met Val Thr AsnGly 195 200 205 gtg ttt gat cgt cac ccc aac cta cag ctc att atg ggt catcta ggt 672 Val Phe Asp Arg His Pro Asn Leu Gln Leu Ile Met Gly His LeuGly 210 215 220 gaa cat gtg ccg ttt gat atg tgg cgc att aat cat tgg ttcgag gac 720 Glu His Val Pro Phe Asp Met Trp Arg Ile Asn His Trp Phe GluAsp 225 230 235 240 cgc aag aag ttg ttg ggg ttg gcg gag acg tgt aag aagacg att cgg 768 Arg Lys Lys Leu Leu Gly Leu Ala Glu Thr Cys Lys Lys ThrIle Arg 245 250 255 gag tac ttt gct cag aat atc tgg att acg act tct gggcac ttt tcg 816 Glu Tyr Phe Ala Gln Asn Ile Trp Ile Thr Thr Ser Gly HisPhe Ser 260 265 270 acg acc acg ttg aac ttc tgc atg gcg gag gtc ggg gtcgat cgc att 864 Thr Thr Thr Leu Asn Phe Cys Met Ala Glu Val Gly Val AspArg Ile 275 280 285 ttg ttc tcg att gat tat ccg ttc gag acg ttt gag gatgcg tgt gtt 912 Leu Phe Ser Ile Asp Tyr Pro Phe Glu Thr Phe Glu Asp AlaCys Val 290 295 300 tgg ttt gat ggc gcg gag ttg aat ctt tcc gat aag gctaag gtc ggg 960 Trp Phe Asp Gly Ala Glu Leu Asn Leu Ser Asp Lys Ala LysVal Gly 305 310 315 320 agg gat aat gcg gcg agg ttg ttt aag ttg ggg gcgttt agg gat tat 1008 Arg Asp Asn Ala Ala Arg Leu Phe Lys Leu Gly Ala PheArg Asp Tyr 325 330 335 gat gcg aag gtt aag gct tag gttgggaactaggattaatg gaatgaaata 1059 Asp Ala Lys Val Lys Ala 340 aatatgactgttttttgaaa aaaaaaaaaa aaaaaaa 1096 <210> SEQ ID NO 2 <211> LENGTH: 342<212> TYPE: PRT <213> ORGANISM: Aspergillus niger <220> FEATURE: <221>NAME/KEY: INIT_MET <222> LOCATION: 1 <221> NAME/KEY: ACT_SITE <222>LOCATION: 251 <300> PUBLICATION INFORMATION: <301> AUTHORS: Santha,Ramakrishnan Dickman, Martin B. O′Leary, Marion H. <302> TITLE:2,3-Dihydroxybenzoic Acid Decarboxylase From Aspergillus niger:Mechanism, Cloning And Overexpression. <303> JOURNAL: Faseb Journal<304> VOLUME: 11 <305> ISSUE: 9 <306> PAGES: A1017 <307> DATE:1997-07-31 <308> DATABASE ACCESSION NUMBER: SWISSPROT Accession no.P80346 (fragments of 2,3-dihydroxybenzoic <309> DATABASE ENTRY DATE:1995-11-01 <400> SEQUENCE: 2 Met Leu Gly Lys Ile Ala Leu Glu Glu Ala PheAla Leu Pro Arg Phe 1 5 10 15 Glu Glu Lys Thr Arg Trp Trp Ala Ser LeuPhe Ser Val Asp Pro Glu 20 25 30 Thr His Val Lys Glu Ile Thr Asp Ile AsnLys Leu Arg Ile Glu His 35 40 45 Ala Asp Lys Tyr Gly Val Gly Tyr Gln IleLeu Ser Tyr Thr Ala Pro 50 55 60 Gly Val Gln Asp Ile Trp Asp Pro Val GluAla Gln Ala Leu Ala Val 65 70 75 80 Glu Ile Asn Asp Tyr Ile Ala Glu GlnIle Arg Asp Lys Pro Asp Arg 85 90 95 Phe Gly Ala Phe Ala Thr Leu Ser MetHis Asn Pro Gln Glu Ala Ala 100 105 110 Ser Glu Leu Arg Arg Cys Val GlnThr Tyr Gly Phe Lys Gly Ala Leu 115 120 125 Val Asn Asp Thr Gln Arg AlaGly Pro Asp Gly Asp Asp Met Ile Phe 130 135 140 Tyr Asp Asn Ala Ser TrpAsp Ile Phe Trp Gln Thr Cys Thr Glu Leu 145 150 155 160 Asp Val Pro LeuTyr Leu His Pro Arg Asn Pro Thr Gly Thr Ile Tyr 165 170 175 Glu Lys LeuTrp Ala Asp Arg Lys Trp Leu Val Gly Pro Pro Leu Ser 180 185 190 Phe AlaGln Gly Val Ser Leu His Val Leu Gly Met Val Thr Asn Gly 195 200 205 ValPhe Asp Arg His Pro Asn Leu Gln Leu Ile Met Gly His Leu Gly 210 215 220Glu His Val Pro Phe Asp Met Trp Arg Ile Asn His Trp Phe Glu Asp 225 230235 240 Arg Lys Lys Leu Leu Gly Leu Ala Glu Thr Cys Lys Lys Thr Ile Arg245 250 255 Glu Tyr Phe Ala Gln Asn Ile Trp Ile Thr Thr Ser Gly His PheSer 260 265 270 Thr Thr Thr Leu Asn Phe Cys Met Ala Glu Val Gly Val AspArg Ile 275 280 285 Leu Phe Ser Ile Asp Tyr Pro Phe Glu Thr Phe Glu AspAla Cys Val 290 295 300 Trp Phe Asp Gly Ala Glu Leu Asn Leu Ser Asp LysAla Lys Val Gly 305 310 315 320 Arg Asp Asn Ala Ala Arg Leu Phe Lys LeuGly Ala Phe Arg Asp Tyr 325 330 335 Asp Ala Lys Val Lys Ala 340 <210>SEQ ID NO 3 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic <400>SEQUENCE: 3 ataagaatgc ggccgctttt tttttttttt 30 <210> SEQ ID NO 4 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 4 ggaattcatgytbggyaaga tcgc 24 <210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic <400> SEQUENCE: 5 aattcatatg ttgggtaaga tcg 23

I claim:
 1. A first isolated polynucleotide that hybridizes to a secondisolated polynucleotide comprising SEQ ID NO: 1 under stringentconditions characterized by hybridization in 6 times SSC or 6 times SSPEand about 40% formamide at about 37° C. and a wash at about 37° C. with2 times SSC or 2 times SSPE containing about 0.1% SDS, wherein the firstisolated polynucleotide encodes a protein that decarboxylates2,3-dihydroxybenzoic acid to form catechol.
 2. A vector comprising afist polynucleotide that hybridizes to a second polynucleotidecomprising SEQ ID NO: 1 under stringent conditions characterized byhybridization in 6 times SSC or 6 times SSPE and about 40% formamide atabout 37° C. and a wash at about 37° C. with 2 times SSC or 2 times SSPEcontaining about 0.1I% SDS, wherein the first polynucleotide encodes aprotein that decarboxylates 2,3-dihydrorybenzoic acid to form catechol.3. The vector of claims wherein the vector contains regulatory elementsoperably linked to the first polynucleotide.
 4. A host cell transformedwith a vector comprising a first polynucleotide that hybridizes to asecond polynucleotide comprising SEQ ID NO: 1 under stringent conditionscharacterized by hybridization in 6 times SSC or 6 times SSPE and about40% formamide at about 37° C. and a wash at about 37° C. with 2 timesSSC or 2 times SSPE containing about 0.1% SDS, wherein the firstpolynucleotide encodes a protein that decarboxylates2,3-dihydroxybenzoic acid to form catechol.
 5. A method for theproduction of an isolated protein that decarboxylates2,3-dihydroxybenzoic acid to form catechol wherein the protein isencoded by a first polynucleotide that hybridizes to a secondpolynucleotide comprising SEQ ID NO: 1 under stringent conditionscharacterized by hybridization in 6 times SSC or 6 times SSPE and about40% formamide at about 37° C. and a wash at about 37° C. with 2 timesSSC or 2 times SSPE containing about 0.1% SDS comprising: a)transforming a host cell with a vector comprising said firstpolynucleotide; b) causing said host cell to,produce the protein; and c)isolating the protein.
 6. A method for the production of a firstisolated polynucleotide that hybridizes to a second polynucleotidecomprising SEQ ID NO:1 under stringent conditions characterized byhybridization in 6 times SSC or 6 times SSPE and about 40% formamide atabout 37° C. and a wash at about 37° C. with 2 times SSC or 2 times SSPEcontaining about 0.1% SDS, wherein the first isolated polynucleotideencodes a protein that decarboxylates 2,3-dihydroxybenzoic acid to formcatechol comprising: a) causing a host cell to produce the firstpolynucleotide; and b) isolating the first polynucleotide.
 7. Apolynucleotide comprising pET22b(+)DHBD.
 8. A host cell comprisingpET22b(+)DHBD.